Single-Strand Conformation Polymorphism Analysis by Microchip Electrophoresis for the Rapid Detection of Point Mutation in Human Obesity Gene

Abstract

We describe an effective method of microchip electrophoresis (ME) based on single strand conformation poly-morphism (SSCP) analysis to rapidly detect the point mutation, Leu72Met, in a human obesity gene. The 207-bp dsDNA in the Leu72Met region, an estimate of the child obesity DNA mutant, was amplified by polymerase chain reaction (PCR) and submitted to a conventional glass microchip analysis with a sieving matrix of 1.75% poly(vinylpyrrolidone) (Mr 1 300 000), 1.0% poly(ethyleneoxide) (Mr 600 000) and 5.0% w/w glycerol. When combined with base stacking (BS) with hydroxide ions, the SSCP-ME provided rapid analysis as well as sensitive detection. The detection sensitivity was effectively enhanced in the OH- concentration range of 0.01-0.025 M NaOH. The sensitivity and speed of this ME-based SSCP methodology for the rapid detection of Leu72Met point mutations makes this an attractive method for diagnosing childhood obesity in a clinical diagnostic laboratory.